Intermolecular disulfide bonds stabilize VirB7 homodimers and VirB7/VirB9 heterodimers during biogenesis of the Agrobacterium tumefaciens T-complex transport apparatus.

Abstract

The Agrobacterium tumefaciens VirB7 lipoprotein contributes to the stabilization of VirB proteins during biogenesis of the putative T-complex transport apparatus. Here, we report that stabilization of VirB7 itself is correlated with its ability to form disulfide cross-linked homodimers via a reactive Cys-24 residue. Three types of f8-mercaptoethanol-dissociable complexes were visualized

Suppression of mutant phenotypes of the Agrobacterium tumefaciens VirB11 ATPase by overproduction of VirB proteins

“…Previous studies in this laboratory established that certain VirB proteins such as VirB7 and VirB9 contribute to the stabilization of other VirB proteins (6,23). Conversely, a transdominant VirB7::PhoA41 hybrid protein was shown to have a destabilizing effect on other VirB proteins, most probably by blocking transporter assembly (6,23,48). We examined the potential for the class II or III VirB11 derivatives to disrupt assembly of VirB proteins into a stabilized structure by comparing levels of VirB proteins in A348(pXZB1xx) merodiploid strains, the isogenic strain A348(pXZB100), and wild-type strain A348.…”

“…Two lines of evidence suggest that these proteins undergo complex formation. First, the VirB7-VirB9 heterodimer has a stabilizing effect on VirB10 and VirB11 (6,23,48). Strains impaired for assembly of the VirB7-VirB9 heterodimer accumulate diminished levels of VirB10 and VirB11, whereas, conversely, strains carrying constructs that allow for the regulated synthesis of the VirB7-VirB9 heterodimer accumulate both VirB10 and VirB11 in rough proportion to the amount of heterodimer.…”

“…Our findings are best interpreted in the context of the following information. Work in our laboratory (23,48) and the laboratories of A. Das (1), P. Zambryski (2), and A. Binns (3) has shown that VirB7 and VirB9 assemble as a covalently cross-linked heterodimer predominantly in the outer membrane. By contrast, VirB10 is a monotopic cytoplasmic membrane protein with a small N-terminal domain in the cytoplasm (22,53) and VirB11 resides exclusively at the cytoplasmic face of the cytoplasmic membrane (22,39).…”

The mating pair formation system of conjugative plasmids—A versatile secretion machinery for transfer of proteins and DNA

“…VirB7 of A. tumefaciens is required for stable expression of a series of Mpf components (VirB4, VirB9, VirB10, and VirB11) and also stabilizes the integrity of the T pilus (Beaupré et al, 1997;Fernandez et al, 1996b;Sagulenko et al, 2001b). By tying intermolecular disulWde bridges, VirB7 is able to form homodimers as well as heterodimers with VirB9, an observation which has been made in multiple T4SS (Anderson et al, 1996;Baron et al, 1997b;Das et al, 1997;Farizo et al, 1996;Fernandez et al, 1996b;Harris et al, 2001;Spudich et al, 1996). VirB9, in turn, is an outer-membrane component of the so-called core complex comprising VirB8, VirB9, and VirB10 (see below).…”

“…Addi- Table 2 Interactions between VirB/VirD4 components a B, detected biochemically (in vitro); G, suggested by genetic arrangement; H, detected by two-hybrid interaction analysis; O, suggested by other indirect evidence. Citations: 1 Spudich et al (1996); 15 Anderson et al (1996); 16 Farizo et al (1996); 17 Harris et al (2001); 18 Das et al (1997); 19 Das and Xie (2000); 20 Kumar and Das (2001); 21 Gilmour et al (2003); 22 Llosa et al (2003); 23 Krause et al (2000a); 24 Yeo et al (2000); 25 Hormaeche et al (2002); 26 Schröder et al (2002); 27 Kumar and Das (2002).…”